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. 2013;9(9):e1003805.
doi: 10.1371/journal.pgen.1003805. Epub 2013 Sep 19.

N-alpha-terminal acetylation of histone H4 regulates arginine methylation and ribosomal DNA silencing

Vassia Schiza  1 , Diego Molina-Serrano, Dimitris Kyriakou, Antonia Hadjiantoniou, Antonis Kirmizis
Affiliations

N-alpha-terminal acetylation of histone H4 regulates arginine methylation and ribosomal DNA silencing

Vassia Schiza et al. PLoS Genet. 2013.

Abstract

Post-translational modifications of histones play a key role in DNA-based processes, like transcription, by modulating chromatin structure. N-terminal acetylation is unique among the numerous histone modifications because it is deposited on the N-alpha amino group of the first residue instead of the side-chain of amino acids. The function of this modification and its interplay with other internal histone marks has not been previously addressed. Here, we identified N-terminal acetylation of H4 (N-acH4) as a novel regulator of arginine methylation and chromatin silencing in Saccharomyces cerevisiae. Lack of the H4 N-alpha acetyltransferase (Nat4) activity results specifically in increased deposition of asymmetric dimethylation of histone H4 arginine 3 (H4R3me2a) and in enhanced ribosomal-DNA silencing. Consistent with this, H4 N-terminal acetylation impairs the activity of the Hmt1 methyltransferase towards H4R3 in vitro. Furthermore, combinatorial loss of N-acH4 with internal histone acetylation at lysines 5, 8 and 12 has a synergistic induction of H4R3me2a deposition and rDNA silencing that leads to a severe growth defect. This defect is completely rescued by mutating arginine 3 to lysine (H4R3K), suggesting that abnormal deposition of a single histone modification, H4R3me2a, can impact on cell growth. Notably, the cross-talk between N-acH4 and H4R3me2a, which regulates rDNA silencing, is induced under calorie restriction conditions. Collectively, these findings unveil a molecular and biological function for H4 N-terminal acetylation, identify its interplay with internal histone modifications, and provide general mechanistic implications for N-alpha-terminal acetylation, one of the most common protein modifications in eukaryotes.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Deletion or inactivation of NAT4 increases the levels of H4R3me2a.
(A) Whole cell extracts from the indicated deletion strains were analyzed by western blotting with an antibody against H4R3me2a (top panel). Equal loading was monitored using an antibody against actin (bottom panel). (B) Whole cell extracts from a wild-type strain (NAT4, lane1) and another one carrying a NAT4 deletion (nat4Δ, lane 2) were analyzed using antibodies against various H4R3 methylation states. Equal loading was monitored with H3 and H4 antibodies. (C) Sequence alignment of the catalytic motifs A and B of the five S. cerevisiae N-alpha acetyltransferases (NATs). The residues that were mutated within each motif to generate the Nat4 catalytic mutants are highlighted in grey. (D) Whole cell extracts from strains containing wild-type NAT4 (lanes 1 and 3)) and different NAT4 mutants (lanes 2, 4, 5 and 6) were analyzed by western blotting using antibodies against H4R3me2a (top panel) and H3 (bottom panel). The strain in lane 2 represents a Nat4 deletion strain. All catalytic mutant strains (lanes 4–6) have a C-terminal hemagglutinin (HA) tag. The strain containing the mutations within motif A is designated as nat4cmA (lane 4), the one with mutations in motif B is nat4cmB (lane 5) and the one with mutations in both motifs is noted as nat4cmAB (lane 6). Their equivalent wild-type strain contains only the C-terminal HA-tag (lane 3).
Figure 2
Figure 2. Deletion of NAT4 enhances silencing and H4R3me2a deposition across the rDNA locus.
(A) Silencing assays for the rDNA region were performed with wild-type (row 1) or nat4Δ (row 2) strains. Both strains (NAT4 and nat4Δ) carry a copy of the URA3 gene, that encodes for an essential enzyme in the Uracil metabolic pathway, inserted in the rDNA locus (RDN1::URA3). This enzyme metabolizes 5′-Fluoroorotic acid (FOA) into a toxic compound, and the ability of the cell to survive in the presence of FOA depends on the degree of silencing in the rDNA region, such that stronger silencing coincides with more cell growth. The cells were spotted in 10-fold dilutions on SC medium (right panel) or SC+FOA (left panel) and grown for 48 h at 30°C. (B) Expression levels of rRNAs 5S, 25S, 5.8S, 18S, 35S and the RPP0 gene were analyzed by qRT-PCR using total RNA extracted from NAT4 and nat4Δ strains. (C) Schematic of the budding yeast rDNA locus on chromosome XII. The rDNA region represents an array consisting of ∼150 tandem copies of a 9.1 kb repeating unit. Each repeat contains the genes RDN5 and RDN37 (encodes the 35S primary transcript) as well as two non-transcribed spacers (NTS1, NTS2), two external transcribed spacers (ETS1, ETS2) and two internal transcribed spacers (ITS1, ITS2). Primers were designed along the rDNA locus as indicated by the red lines and letters A–N. ChIP experiments were performed in the NAT4 and nat4Δ strains using antibodies against H4R3me2a (top panel) and N-acH4 (bottom panel). The immunoprecipitated chromatin was analyzed by qRT-PCR using the primers A–N. (see Table S2 for their sequence). The enrichment from each antibody was normalized to the levels of histone H4. Error bars in (B) and (C) indicate s.e.m for duplicate experiments.
Figure 3
Figure 3. Nat4 inhibits rDNA silencing and H4R3me2a through N-terminal acetylation of H4.
(A) Whole cell extracts prepared from the wild-type strains (H4WT and H2AWT) and their correspondent Serine-to-Alanine mutants in position 1 (H4S1A and H2AS1A) were analyzed by western blotting using antibodies against H4R3me2a (top panel) and H3 as control (bottom panel). (B) ChIP experiments were performed in the same strains as in (A) using the H4 and H4R3me2a antibodies. The immunoprecipitated chromatin was analyzed by qRT-PCR using primer I specific to the RDN25 gene (shown in figure 2C). The enrichment from each antibody was normalized to 1% of the total input DNA. (C) Gene expression analysis of 25S rRNA performed using the same strains as in (A). The expression levels of the 25S rRNA were normalized to the levels of RPP0. (D) Silencing assays for the rDNA locus were performed with a wild-type (H4WT) or a H4S1A mutant strain as described in (2A). (E) Whole yeast cell extracts prepared from the wild-type strains NAT4 (lane 1), and the mutant strains nat4Δ (lane 2) and nat4Δ/hNAA40 (that carries a NAT4 deletion and a plasmid that expresses ectopically hNAA40, lane 3) were analyzed by western blotting using the indicated antibodies. Equal loading was monitored by an H3 antibody (top panel). (F) ChIP experiments performed in the indicated strains as in (E) using antibodies against H4R3me2a and N-acH4. The enrichment of each antibody was normalized to the levels of H4 occupancy. Error bars in (B), (C) and (F) indicate s.e.m for duplicate experiments.
Figure 4
Figure 4. N-acH4 inhibits the Hmt1 methyltransferase activity towards H4R3.
(A) In vitro methylation assays were performed with synthetic biotinylated peptides representing the first 20 amino acids of histone H4 in the absence (lanes 1 to 9) or presence (lanes 10 to 14) of purified yeast Hmt1. The methyltransferase activity was monitored by western blotting using an antibody against H4R3me2a. Peptide loading was controlled by ponceau staining. (B) ChIP experiments were performed in the wild-type (H4WT NAT4) and the mutant strains carrying a NAT4 deletion (H4WT nat4Δ), an H4 Arginine-to-Lysine mutation in position 3 (H4R3K NAT4) or both (H4R3K nat4Δ), using antibodies against H4, H4R3me2a and N-acH4. The enrichment at RDN25 was analyzed as in (3B). (C) 25S rRNA expression level analysis was performed in the same strains as in (B). The qRT-PCR analysis was performed as in (3C). Error bars in (B) and (C) indicate s.e.m for duplicate experiments.
Figure 5
Figure 5. N-acH4 acts synergistically with H4K5, 8, 12 acetylation to control rDNA silencing, H4R3me2a and cell growth.
(A) Whole yeast cell extracts were prepared from the wild-type (H4WT NAT4) and the mutant strains carrying a NAT4 deletion (H4WT nat4Δ), a triple Lysine-to-Arginine mutation in H4 in positions 5, 8 and 12 (H4K5,8,12R NAT4) or both (H4K5,8,12R nat4Δ) and then analyzed by western blotting using the H4 modification antibodies are shown. Equal loading was monitored with an H3 antibody (bottom panel). (B) ChIP experiments were performed in the same strains as in (A) using the antibodies against H4 and H4R3me2a. The enrichment of each antibody was analyzed as in (3B). (C) 25S rRNA expression level analysis was performed in the wild-type (H4WT NAT4) and the mutant strains carrying a NAT4 deletion (H4WT nat4Δ), a triple H4 Lysine-to-Arginine mutation in positions 5, 8 and 12 (H4K5,8,12R NAT4) both (H4K5,8,12R nat4Δ), and a multiple H4 mutant with a triple Lysine-to-Arginine substitution in positions 5, 8 and 12, an Arginine-to-Lysine mutation in position 3, and a NAT4 deletion (H4Κ5,8,12R H4R3K nat4Δ). The analysis was performed as in (3C). Error bars in (B) and (C) indicate s.e.m for duplicate experiments. (D) Growth assay of the same yeast strains as in (C) plus a multiple H4 mutant with a triple Lysine-to-Arginine substitution in positions 5, 8 and 12, and Serine-to-Alanine mutation in position 1 (H4K5,8,12R H4S1A NAT4). Cells were spotted in 10-fold dilutions on YPAD medium plates. Cell growth was examined at 30°C (left panel) or 37°C (right panel).
Figure 6
Figure 6. Calorie restriction increases RDN25 silencing and the H4R3me2a: NacH4 enrichment ratio.
(A) The levels of 25S rRNA were analyzed by qRT-PCR using total RNA extracted from a wild-type strain (BY4741) grown in minimal media containing different glucose concentrations (2%, 0.5%, 0.1% and 0.05%). (B) ChIP experiments were performed in a wild-type BY4741 strain grown in the same conditions as in (A), using antibodies against H4R3me2a and N-acH4. Their enrichment is normalized to histone H4 and represented as ratio of H4R3me2a to N-acH4. Error bars in (A) and (B) indicate s.e.m for duplicate experiments.
Figure 7
Figure 7. Model depicting the role of N-acH4 in rDNA silencing.
When rDNA expression is required, Nat4-catalyzed H4 N-terminal acetylation inhibits Hmt1-mediated H4R3me2a. Under conditions where rDNA expression needs to be repressed, as in an environment with low glucose concentration, N-terminal acetylation decreases by a mechanism still unknown. This mechanism, that can be active (by an enzyme) or passive (by histone dilution) reduces the levels of N-acH4 and allows Hmt1 to asymmetrically dimethylate H4R3, triggering rDNA silencing. Lysine acetylation on residues 5, 8 and 12 fine-tunes the levels of H4R3me2a, as excessive deposition of this mark leads to a severe growth defect at 30°C or even cell lethality at a higher temperature (37°C).
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Grants and funding

This work was supported by grants from the European Research Council (ERC-2010-Stg, N.260797, ChromatinModWeb) and the Cyprus Research Promotion Foundation (Health/Bio/0609(BE)/09). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.